CURCUMIN AND BETA-SITOSTEROL LOADED HYDROGEL SCAFFOLDS FOR ACCELERATED WOUND HEALING

Abstract

The present invention relates to the development and characterization of curcumin and beta-sitosterol-loaded hydrogel scaffolds aimed at accelerating wound healing. The formulated hydrogel scaffolds employ chitosan as a biopolymer matrix, combined with a co-loading of curcumin and beta-sitosterol. Three distinct formulations were created: a co-loaded hydrogel (curcumin + beta-sitosterol), a curcumin-only hydrogel, and a beta-sitosterol-only hydrogel. Each scaffold underwent evaluation, including Fourier Transform Infrared (FTIR) analysis for chemical characterization, drug content estimation, porosity, water vapor transmission rate, in vitro degradation, and swelling ratio assessment. In vitro drug release studies were conducted using the dialysis bag method. The in vivo wound healing efficacy was further examined using an excision wound model on Sprague Dawley rats, demonstrating superior wound contraction and faster epithelialization in the co-loaded scaffold group. Additionally, a skin irritation study confirmed the biocompatibility of the scaffolds.

Specification

Description:FIELD OF INVENTION
The present invention relates to hydrogel scaffolds loaded with curcumin and beta-sitosterol, designed to accelerate wound healing. This invention addresses enhanced bioavailability and controlled release of therapeutic agents to promote tissue regeneration.
BACKGROUND OF THE INVENTION
In wound healing, effective management is often hindered by inadequate drug delivery, limited bioavailability, and prolonged inflammation, which can delay recovery and increase the risk of infection. Traditional wound dressings are insufficient to provide controlled release of bioactive compounds and may not offer the necessary therapeutic benefits for rapid wound repair. As a result, there is a pressing need for an advanced material that can both protect the wound and deliver healing agents in a controlled and sustained manner.
Curcumin and beta-sitosterol are two bioactive compounds known for their anti-inflammatory, antimicrobial, and antioxidant properties, which are essential for effective wound healing. However, their direct application is limited due to curcumin's low solubility and poor stability and beta-sitosterol's difficulty in penetrating the skin barrier. These limitations restrict the therapeutic efficacy of these agents when applied topically, necessitating a delivery system that can enhance their bioavailability and ensure targeted, sustained release to the wound site.
Hydrogel scaffolds offer an ideal solution to address these challenges. Hydrogels are biocompatible, can retain significant amounts of moisture, and support controlled drug release, making them suitable for wound dressing applications. By incorporating curcumin and beta-sitosterol into a hydrogel matrix, it is possible to overcome their bioavailability issues and provide a scaffold that promotes accelerated wound healing through localized delivery. This approach ensures prolonged release of the active compounds, creating an optimal environment for cellular regeneration and reducing inflammation at the wound site.
IN 202311004358: The present invention is generally directed to a Carbopol hydrogel formulation loaded with herbal nanoparticles for treating chronic diabetic wounds. The invention provides herbal nanoparticles comprising blending of curcumin and gallic acid with the polymer via desolvation method. Different formulations of the hydrogel were prepared that is F1, F2, F3, F4, F5, and F6. nanoparticles formulation in Carbopol hydrogel wherein the Carbopol is present in an amount 0.25g, 0.5g, 1.25g. The percent drug release of curcumin and gallic acid was found to be 89.51% and 85.63%, respectively in 8 hrs. The encapsulation efficiency found to be 71% for curcumin and 85.5% for gallic acid. The percentage yield of nanoparticle formulation was 94%. The invention shows 76.31% curcumin and 69.39% gallic acid release in 9 hours. The invention also provides the drug release for upto 12 hours. The present invention is differ from this as it relates to hydrogel scaffolds loaded with curcumin and beta-sitosterol, designed to accelerate wound healing. This invention addresses enhanced bioavailability and controlled release of therapeutic agents to promote tissue regeneration.
OBJECTS OF THE INVENTION
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative
An object of the present disclosure relates to bioactive hydrogel scaffolds for wound healing.
Another object of the present disclosure is to enhance wound healing efficiency through a synergistic combination of curcumin and ß-sitosterol.
Still another object of the present disclosure is to provide sustained release of bioactive agents for prolonged therapeutic effects.
Another object of the present disclosure is to minimize inflammation by utilizing the anti-inflammatory properties of curcumin.
Still another object of the present disclosure is to support tissue regeneration and epithelialization for faster wound recovery.
Still another object of the present disclosure is to maintain a balanced moisture level for optimal wound healing conditions.
Yet another object of the present disclosure is to ensure high porosity for improved fluid absorption and nutrient exchange.
Yet another object of the present disclosure is to create a biocompatible, biodegradable scaffold for natural degradation post-healing.
Yet another object of the present disclosure is to reduce oxidative stress, promoting a healthy healing environment.
Yet another object of the present disclosure is to provide that the developed scaffolds can be used for various types of wounds, including chronic wounds and surgical incision, and diabetic wounds.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.

SUMMARY OF THE INVENTION
The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

The present invention is generally directed to a bioactive hydrogel scaffold incorporating curcumin and ß-sitosterol in a chitosan matrix, designed to enhance wound healing through controlled drug release. It offers improved tissue regeneration, anti-inflammatory effects, and skin repair, suitable for chronic and surgical wounds.
An embodiment of the present invention the bioactive hydrogel scaffold is composed of curcumin and ß-sitosterol incorporated into a chitosan-based polymer matrix, designed to accelerate wound healing and tissue regeneration. The scaffold is available in three formulation variants: co-loaded (F1), curcumin-loaded (F2), and ß-sitosterol-loaded (F3), each providing distinct therapeutic benefits.
Another embodiment of the invention consists of the following composition: Curcumin (25-50 mg) providing anti-inflammatory and antioxidant benefits, and ß-sitosterol (50-100 mg) contributing to skin repair and wound healing. Chitosan (1.45% w/v), a biocompatible and biodegradable polymer, is dissolved in acetic acid (2% v/v) to form a stable polymer solution, serving as the scaffold's base.
Yet another embodiment of the invention is the preparation process involves dissolving chitosan (1.45% w/v) in acetic acid (2% v/v) under magnetic stirring at 500 rpm to form a homogeneous solution. The required amounts of curcumin and ß-sitosterol are then dispersed into 10 g of the gel precursor, ensuring even drug distribution. The mixture is stirred magnetically until fully homogeneous, incorporating the drugs uniformly within the hydrogel matrix. The resulting mixture is poured into petri dishes and frozen at -50°C for 20 hours to solidify the scaffold structure. The frozen hydrogel is then freeze-dried at -50°C for 24 hours, creating a porous and stable structure that facilitates effective drug release.
Yet another embodiment of the invention is the hydrogel is thoroughly characterized through FTIR analysis, drug content assessment, porosity and swelling ratio tests, and in-vitro degradation studies. Drug release profiles are evaluated using a dialysis bag method to simulate wound conditions. Additionally, skin irritation studies confirm biocompatibility, while in-vivo studies on assess wound healing efficacy through parameters such as wound contraction, healing time, and epithelialization rate.
Yet another embodiment of the invention is results shows that the prepared scaffolds had shown the improved tissue regeneration, anti-inflammatory effects, and skin repair, suitable for chronic and surgical wounds.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig 1: Hydrogel scaffold formulations
Fig 2. FTIR analysis of hydrogel scaffolds
Fig 3. Weight loss during the degradation of hydrogel scaffolds.
Fig 4. Swelling ratio of hydrogel scaffolds
Fig 5. Cumulative percentage drug release from hydrogel scaffolds formulations
Fig 6. Photographic representation of skin irritation study
Fig 7. Photographic representation of wound healing.
Fig 8. Epithelialization period.
Fig 9. Morphological images of histopathological findings at 10x H&E staining: (a) Co-loaded (b) Cu-loaded (c) BS-loaded (d) PC (e) NC

DETAILED DESCRIPTION OF THE INVENTION
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
The present invention relates to a bioactive hydrogel scaffold incorporating curcumin and ß-sitosterol in a chitosan matrix, designed to enhance wound healing through controlled drug release.

The hydrogel scaffold is composed of key components including active pharmaceutical ingredients (APIs) such as curcumin (25-50 mg) for its anti-inflammatory and antioxidant effects, and beta-sitosterol (50-100 mg) for its wound-healing and skin-repairing properties. The polymer base consists of chitosan (1.45% w/v) dissolved in acetic acid (2% v/v) to form a stable polymer solution. The scaffold is formulated in three variants: F1 (Co-loaded) containing 25 mg of curcumin and 50 mg of beta-sitosterol, F2 (Cu-loaded) containing 50 mg of curcumin only, and F3 (BS-loaded) containing 100 mg of beta-sitosterol only.
EXAMPLE 1: Preparation of hydrogel-based scaffold
Chitosan (1.45% w/v) was dissolved in acetic-acid (2% v/v) under magnetic stirring at 500 rpm to prepare a polymer solution. To fabricate the hybrid system, the drug solution was initially dispersed into 10g of the gel precursor. The mixture was then be magnetically stirred to become completely homogeneous. Afterward, hydrogel was poured in petri dishes, followed by freezing for at -50°C for 20 hours. The hydrogel was eventually freeze-dried at -50?C for 24h. By using the optimized procedure three formulation batches were prepared which were shown in table 1.
Table 1: Formulation composition
Formulation Code Formulation batch API Chitosan
F1
Co-loaded
Curcumin (25mg) + ß-sitosterol (50mg)
1.45 % w/v
F2
Cu-loaded Curcumin (50 mg )
1.45 % w/v
F3
BS-loaded ß-sitosterol (100 mg)
1.45 % w/v
The Curcumin and ß-Sitosterol loaded hydrogel-based scaffolds were prepared by lyophilization method using freeze dryer.
EXAMPLE 2: FTIR Analysis
The chemical characterization of the fabricated hydrogel scaffolds was performed using Fourier Transform Infrared Spectroscopy (FTIR). A 1 mm³ sample of each scaffold (N = 1) was analyzed, and the FTIR spectra were recorded using a PE Spectrum 100 FTIR spectrometer (PerkinElmer, Waltham, MA, USA) in the wavelength range of 700-4000 cm?¹. The obtained absorbance peaks were analyzed to identify the chemical structures of the components and any changes that may have occurred due to the scaffold fabrication and crosslinking process.
Results shows that the FTIR spectra, illustrated in Figure 2, were obtained to identify the characteristic bands of functional groups present in the hydrogel components. The FTIR spectrum of pure curcumin displays key absorption bands that highlight its functional groups, including a broad O-H stretching band around 3500-3200 cm?¹, a C=O stretching peak at 1627 cm?¹ characteristic of its a, ß-unsaturated diketone structure, and a C=C stretching peak around 1600 cm?¹ in the aromatic ring. Additionally, peaks between 1026-1275 cm?¹ correspond to the C-O stretching vibrations of the phenolic group, collectively defining curcumin's chemical structure.
For ß-sitosterol, the FTIR spectrum shows a prominent O-H stretching band around 3400-3300 cm?¹, a series of C-H stretching vibrations between 2900-2800 cm?¹, typical of sterol compounds, and a C-O stretching peak between 1050-1150 cm?¹ associated with its alcohol group, indicating the key functional groups in the sterol structure.
The FTIR spectrum of the physical mixture displays characteristic peaks from curcumin, ß-sitosterol, and chitosan, with slight shifts or changes in intensity, suggesting minor physical interactions but no major chemical reaction between them. The O-H stretch from both curcumin and ß-sitosterol remains around 3400 cm?¹, while chitosan's amine absorption appears between 3200-3500 cm?¹, indicating retention of chemical integrity within the mixture.
In the co-loaded hydrogel scaffold, the FTIR spectrum reveals broadened and shifted O-H stretching around 3400 cm?¹, suggesting strong hydrogen bonding among curcumin, ß-sitosterol, and the hydrogel matrix. The attenuation or shift of the C=O stretch (1627 cm?¹) and other key peaks indicates successful encapsulation and interactions between the hydrogel matrix and loaded compounds, confirming integration and possible chemical interactions within the scaffold.
EXAMPLE 3: Drug Content
The drug content of the fabricated hydrogel scaffolds was calculated to assess the efficiency of drug loading. The hydrogel scaffolds were immersed in 100 mL of phosphate buffer saline (PBS) at pH 6.8 and kept overnight to allow the release of the loaded drugs. After 24 hr, 3 mL aliquots were withdrawn, filtered using a Millipore® filter (0.22 µm), and analyzed for drug content using a UV-Visible spectrophotometer. The absorbance was measured at the ?max of 429 nm for curcumin and 228 nm for ß-sitosterol.The drug content was calculated using the following equation:
% Drug Content= (Actual drug content)/(Theoretical drug content)×100 (Eq. 1)
Drug content analysis results was shown in Table 3, which shows that the high encapsulation efficiency in the hydrogel scaffolds for both curcumin (Cu) and ß-sitosterol (BS), either individually or combined. The co-loaded scaffold (Cu+BS) achieved a high drug content of 91±3.8%, indicating effective incorporation of both agents. For single-drug scaffolds, curcumin-loaded hydrogels showed a drug content of 83.73±4.1%, suggesting slightly lower encapsulation, possibly due to curcumin's individual interactions with the hydrogel matrix. Meanwhile, the ß-sitosterol-loaded scaffold demonstrated a drug content of 87.63±2.5%, which was higher than curcumin alone but still less than the co-loaded scaffold, highlighting improved entrapment in the combined formulation.
Table 3. Drug content, Porosity and Water vapor transmission rate of hydrogel scaffolds
S. No. Sample Drug Content (%) Porosity (%) WVTR (g/m²h)
1 Co-loaded 88.46 ± 1.7 (Cu)
91.23 ± 3.8 (BS) 76.73 ± 2.1 69.93 ± 1.2
2 Cu-loaded 83.73 ± 4.1 63.63 ± 1.4 51.594 ± 2.4
3 BS-loaded 87.63 ± 2.5 75.34 ± 3.2 62.412 ± 1.9
EXAMPLE 4: Porosity
The porosity of the scaffolds was determined using the liquid displacement method, with ethanol serving as the displacement liquid. Initially, the scaffold's weight (denoted as 'w') was recorded. The scaffold was then immersed in 5 mL of ethanol, and the initial ethanol volume (V1) was measured. After allowing the scaffold to sit in the ethanol for five minutes, the remaining ethanol volume in the scaffold was noted as V2. The ethanol volume left after removing the scaffold was recorded as V3. The scaffold's porosity was then calculated based on these measurements using the following formula:
% Porosity of the fiber = "V1 - V3 " /"V2-V3" " ×100" (Eq. 2)
The results of the porosity was shown in table 3. Results clearly shows that the co-loaded scaffold (curcumin and ß-sitosterol) had the highest porosity at 76.73%, followed by the ß-sitosterol-loaded scaffold (75%), while the curcumin-loaded scaffold had the lowest porosity (63.63%). The higher porosity in co-loaded and ß-sitosterol scaffolds suggests improved drug loading, enhanced surface area, and better fluid diffusion, supporting efficient, sustained drug release. The lower porosity in the curcumin-loaded scaffold indicates a denser matrix, likely due to stronger curcumin-hydrogel interactions, potentially affecting fluid diffusion and drug release.
EXAMPLE 5: Water vapor transmission rate
The ability of skin scaffolds to regulate water vapor transmission rate (WVTR) plays a crucial role in maintaining a moist wound environment and preventing excessive fluid loss, particularly in burn wounds. An optimal WVTR promotes faster wound healing by preventing dehydration while avoiding the accumulation of wound exudates, which can lead to infection. A bottle with a mouth diameter of 35 mm containing 10 mL of water was used for the experiment. A scaffold sample, 40 mm in diameter, was placed over the bottle's mouth to act as a cap. The setup was placed in an oven maintained at 35°C for 24 hours. The WVTR was then calculated based on the weight loss of the bottle, attributed to water vapor transfer. The following formula was applied to determine WVTR:
WVTR=((Wi-Wt))/(A ×24)×10^6 g/m^2 h (Eq. 3)
where "A" represents the area of the bottle's mouth (in mm²), and the WVTR is expressed in g/m² h. Wi and Wt refer to the initial and final weights of the bottle, before and after the oven exposure, respectively.
Results shows that the WVTRs of the fabricated co-loaded, curcumin-loaded (Cu-loaded), and ß-sitosterol-loaded (BS-loaded) hydrogel scaffolds were measured., and shown in table 3. The results showed that the WVTR values for the Co-loaded, Cu-loaded, and BS-loaded scaffolds were 69.93 g/m²h, 51.594 g/m²h, and 62.412 g/m²h, respectively. These values fall within a suitable range for wound healing applications when compared to commercialized wound dressings, where WVTR ranges from 33 g/m²h (OpSite) to 208 g/m²h (Omiderm). This suggests that the fabricated scaffolds have appropriate WVTR values to support the healing process High WVTR values can lead to excessive drying of the wound bed, which may delay healing, while lower WVTR values can cause the accumulation of wound exudates, increasing the risk of bacterial infection. The WVTR values of the scaffolds developed in this study strike a balance between preventing dehydration and maintaining a moist wound environment, making them suitable candidates for wound healing, particularly in the case of burn wounds where fluid loss is a major concern.
EXAMPLE 6: In-vitro degradation study
An in vitro degradation study was conducted over a 42-day period, with measurements taken on days 7, 14, 21, and 42. Scaffolds were cut into 1 x 1 cm² pieces, and the initial weight (W0) of each piece was recorded before immersion in 3 mL of phosphate buffer saline (PBS) at pH 6.8. At predetermined intervals, the samples were removed from the PBS, dried at 45°C for 24 hours, and then reweighed (Wi) [Shahmohammadi et al., 2024]. All experiments were performed in triplicate. The percentage of weight loss was calculated using the following formula:
Weight loss(%)= ((W0 - Wi)/W0 )×100 (Eq. 4)
The fabricated hydrogel scaffolds were designed for gradual auto-degradation, directly impacting the release profiles of the loaded drugs. To assess stability and degradation, we measured percentage weight loss over four weeks (Figure 3). Results showed progressive weight loss across all hydrogel types, with the co-loaded scaffold (curcumin and ß-sitosterol) exhibiting the highest degradation rate, reaching 28.57% weight loss by week 4. This increased degradation likely results from the scaffold's reduced rigidity and lower structural stability.
In comparison, the single-loaded curcumin and ß-sitosterol hydrogels had slightly lower degradation rates, with final weight losses of 23.01% and 25.53%, respectively. The co-loaded scaffold's rapid degradation, possibly due to interactions between both active agents and the hydrogel matrix, may facilitate a faster drug release than the more stable single-loaded scaffolds.
Results clearly indicate that the co-loaded scaffold, being less rigid, displayed the most rapid degradation, potentially leading to faster drug release profiles compared to the more stable Cu-loaded and BS-loaded formulations. These results suggest that the degradation behavior of the hydrogels can be fine-tuned by adjusting the loading composition, which in turn can control the drug release rate to match the desired therapeutic requirements.
EXAMPLE 7: Swelling ratio
The water absorption capacity of the samples was evaluated by measuring their weight immediately after freeze-drying (Wd). The samples were then submerged in phosphate-buffered saline (PBS) at room temperature. At specific intervals, their wet weights (Ww) were recorded, ensuring any excess surface water was removed using filter paper. The swelling ratio was determined using the following equation [Venkatesan et al., 2015]:
% Swelling ratio=(W_w-W_d)/W_d ×100 (Eq. 5)
The swelling test results showed a time-dependent increase in the swelling ratio of chitosan-containing hydrogel scaffolds, a key factor affecting fluid absorption and drug release. As illustrated in Figure 4, all formulations-co-loaded, Cu-loaded, and BS-loaded-displayed a steady rise in swelling ratio over a 6-hour period. The co-loaded scaffold with both curcumin and ß-sitosterol exhibited the highest swelling ratio, suggesting that the presence of both agents in the chitosan matrix enhances fluid uptake due to the scaffold's hydrophilic nature.
Chitosan's biocompatibility and swelling capability in aqueous environments support this trend, as the scaffold maintains structural integrity during hydration. The swelling ratio is directly linked to the release mechanism; higher swelling facilitates faster drug release by promoting diffusion from the expanded matrix. Consequently, the increased swelling in the co-loaded scaffold indicates its potential for a more controlled, sustained release of curcumin and ß-sitosterol, compared to the single-loaded formulations.
EXAMPLE 8: In-vitro drug release study
The release profiles of curcumin and ß-sitosterol from the hydrogel scaffolds were examined using the dialysis bag method. This was compared to the release profiles of curcumin and ß-sitosterol individually in PBS (pH 6.8). Each hydrogel scaffold, containing 10 mg of drug, was placed into a pre-soaked dialysis cellulose bag filled with 1 mL of PBS. The bag was sealed and immersed in 100 mL of PBS (acting as the release medium) in a 200 mL glass beaker, ensuring sink conditions. The setup was maintained on a magnetic stirrer at 50 rpm and 37 ± 0.2°C. Samples of 2 mL were taken at regular intervals to determine drug concentration using a UV spectrophotometer, with an equivalent volume of fresh PBS added to maintain the release conditions.The cumulative release from the co-loaded hydrogel scaffolds, as well as those loaded with either curcumin or ß-sitosterol, was plotted against time.
The in-vitro drug release profile of curcumin (Cu), ß-sitosterol (BS), and their co-loaded formulations was evaluated over a 12-hr period, as shown in Figure 5. The cumulative percentage of drug release was monitored for each formulation, and notable differences in the release patterns were observed. All formulations exhibited a biphasic release pattern, characterized by an initial burst release followed by a more sustained release phase. The initial burst release can be attributed to the release of drug molecules that were either loosely bound or located on the surface of the scaffold. This was followed by a more controlled release phase, likely driven by the diffusion of the drugs through the hydrogel matrix.
Among the formulations, the co-loaded (Cu and BS) scaffold demonstrated the highest cumulative drug release, reaching approximately 30% after 12 hrs. This enhanced release may be due to synergistic interactions between curcumin and ß-sitosterol within the scaffold, facilitating their diffusion through the hydrogel network. The co-loaded formulation appears to promote a more sustained release compared to the single-drug formulations, which can be advantageous for applications requiring prolonged therapeutic effects. In comparison, the curcumin-only and ß-sitosterol-only formulations displayed relatively slower release rates, with the curcumin-loaded scaffold showing a slightly higher cumulative release than the ß-sitosterol formulation. This difference in release rates could be attributed to the molecular size and solubility of the drugs, as well as their interaction with the hydrogel matrix. Curcumin, being more hydrophobic, may interact more strongly with the hydrogel, resulting in a slower release compared to ß-sitosterol.
EXAMPLE 9 : Skin irritation study
To assess skin irritation, white male albino rats, weighing between 160-200 g were divided into three groups (n=2). Depilatories were used to remove hair from the backs of the animals, and a 4cm³ area was marked on each side. One side was considered a control whereas the other was experimental. After hair depletion (wait for 24 h) formulation was applied (10 mg/rats) daily for 7 days and observation was made for any sensitivity and the reaction if any was graded as:
A - No reaction, B - Slight, patchy erythema, C - Slight but confluent or moderate but patchy erythema, D - Moderate erythema, E - Severe erythema with or without edema.
As presented in Figure 6, all formulations showed "A" results (No reaction) across the seven days of observation. This indicates that none of the animals exhibited visible signs of irritation, redness, swelling, or any adverse reactions throughout the testing period. The findings confirm the excellent biocompatibility of the formulations. The developed hydrogel scaffolds are non-irritating and safe for topical application.
EXAMPLE 10: Wound healing activity
Female Sprague Dawley rats weighing between 160-200 gm were procured Animals were caged in polypropylene cages each containing a maximum of five animals. They were housed in the Departmental Animal House of Faculty of Pharmacy, Integral University under controlled conditions of temperature 23°C ± 5°C, relative humidity of 50%±20%, and with a 12-hr light-dark cycle. The animals were provided with a standard pellet diet and had unrestricted access to drinking water.
Excision wound model
The excisional wound was created by cutting a way of 2×2 cm2 area of skin of rats from the predetermine area. After anesthetizing the rats via injection thiopentone sodium 40mg/kg body weight), their dorsal hair was shaved. The wound site was decontaminated, employing 10% povidone-iodine. The simple wound was created by cutting a way of 2×2 cm2 area of skin of rats from the predetermine area. The Sprague Dawley (SD) rats were randomly divided into five groups each consisting of four animals. Study was conducted as per the protocol given in Table 2. Wounds were left air-exposed and the wound healing ratio was evaluated on days 0, 5, 10, 15, and 21 of the experiment. Complete wound healing was considered as a percentage of wound contraction and epithelialization time.
Table 4. Study design
Groups
(n=4) No of animals Dosage, Route of Administration and Duration
I - Negative Control (NC) 4 Nothing was applied on wound for 21 days
II - Standard Positive Control (PC) 4 Standard dose of Megaheal (0.02 % w/w) was applied topically (2×2 cm2) on wound for 21 days
III - (Test 1: Co-loaded) 4 0.75 % w/w Co-loaded hydrogel scaffold topically (2×2 cm2) on wound for 21 days
IV - (Test 2; Cu-loaded) 4 0.50% w/w Cu-loaded hydrogel scaffold topically (2×2 cm2) on wound for 21 days
V - (Test 3: BS-loaded) 4 1% w/w BS-loaded hydrogel scaffold topically (2×2 cm2) on wound for 21 days

Assessment of healing process
Healing was analyzed on basis of wound contraction & epithelialization. All the groups were inspected every alternate day until epithelization & wound closure was recorded. Wound contraction was analyzed as the percentage of the initial wound size.
Wound contraction
After wound creation, the excision wound margins were traced by the changes in wound area planimetrically. The area of the wound was measured by the Vernier calliper on days 0th, 7th, 14th, and 21st. It was compared with the size of the relevant wound at day 6. From the tracings, the wound surface area was evaluated [Bhutta et al., 2021]. To determine the wound contraction (%), the wound region on day 0 was taken as 100%. The percentage of wound contraction was determined using the following formula:
Wound contraction ratio ( % ) = (( A 0 -A t ))/(A 0)×100 (Eq. 6)
A0 is the wound area on day 0, and At is the wound area at the specified time point.
Epithelialization
The total time taken for the epithelialization was also measured in days. This represents the number of days taken for complete healing. The epithelization period was monitored by noting the number of days required for leaving no raw wound behind. On days 0, 7, 14, and 21st of the experiment, lesions were photographed with a digital camera. The lesion area was obtained from the pictures and was used to calculate the percentage of wound area.
Healing time
Healing time was considered from the day of wound induction until re-epithelisation. It was estimated by summarizing observations until the scar fell off.
The wound healing activity of the developed hydrogel scaffold formulation was assessed using the excision wound model. Figure 7 illustrates the photographic representation of the wound healing process. The application of the co-loaded hydrogel scaffold (curcumin and ß-sitosterol) for 7 days demonstrated accelerated wound healing and a higher percentage of wound contraction compared to the individual Cu-loaded and BS-loaded formulations, as well as the control groups.
Wound Contraction
As seen in Table 4, the co-loaded hydrogel scaffold achieved approximately 95% wound closure by day 15 and nearly 100% by day 17. In contrast, the Cu-loaded and BS-loaded formulations displayed 91.5% and 92.5% wound closure by day 15, respectively, reaching almost complete closure by day 19. The positive control group (treated with Megaheal gel) showed slower wound healing, achieving 81% wound closure by day 15 and 95% by day 21, while the negative control group displayed the slowest rate of wound healing, with only 62% wound closure by day 15 and 84% by day 21.
The superior healing performance of the co-loaded hydrogel scaffold can be attributed to the combined therapeutic effects of curcumin and ß-sitosterol, both of which possess anti-inflammatory and antioxidant properties that enhance tissue regeneration and collagen synthesis. The accelerated rate of wound closure observed in the co-loaded group suggests that the formulation effectively promotes the proliferation of fibroblasts and keratinocytes, essential for wound healing.
Epithelialization Period
In terms of epithelialization, wounds treated with the co-loaded hydrogel scaffold exhibited faster regeneration. Epithelialization was initiated on day 19 in the co-loaded group, compared to day 21 in both the Cu-loaded and BS-loaded groups. This earlier onset of epithelialization indicates improved re-epithelialization and enhanced tissue repair. In comparison, the positive control group-initiated epithelialization by day 25, and the negative control group showed epithelialization only by day 30 (Figure 8).
Table 5. Wound healing assessment
S. No.
Post-wound day % Wound contraction
Co-loaded Cu-loaded BS-loaded PC
(Megaheal) NC
0 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0
3 15±1.5 7.5±1.3 5±2.1 6.50±1.2 4±1.1
6 29±2.2 19.5±1.3 22.5±1.2 14.5±1.1 12.5±1.2
9 39.5±2.3 33.5±2.1 32.5±1.4 19.5±0.8 15.5±1.3
12 80.5±1.8 77.5±1.6 78±1.8 63.5±1.2 41±2.4
15 95±1.2 91.5±1.2 92.5±1.1 81±2.1 62±1.4
18 100±0.6 96±1.6 97±1.4 85±1.2 71±1.6
21 100±0.00 100±0.00 100±0.00 95±1.4 84±1.6

Experiment 10: Histopathological assessment
On the 21th day, granulation tissue was collected for histopathological evaluation. The tissue was cut into 5-10 µm sections using a microtome and stained with Hematoxylin and Eosin (H&E). Stained tissue slides were examined and photographs were captured using a camera
Figure 9 shows the histopathological impact of different treatments on wound healing across various groups: co-loaded (curcumin and ß-sitosterol), curcumin-loaded (Cu-loaded), ß-sitosterol-loaded (BS-loaded), positive control, and negative control. The co-loaded group exhibits well-organized tissue, advanced epithelialization, and robust granulation tissue with minimal inflammation, indicating enhanced healing through a synergistic effect between curcumin and ß-sitosterol.
The Cu-loaded group demonstrates moderate tissue regeneration with some remaining inflammation and lower fibroblast activity, suggesting curcumin's antioxidant and anti-inflammatory effects support healing but are less effective alone. The BS-loaded group shows less organized tissue, persistent inflammation, and slower granulation tissue formation, implying that ß-sitosterol is less effective on its own for rapid healing.
The positive control has moderate healing with some organization but lacks the accelerated recovery seen in the co-loaded group, while the negative control shows delayed healing, marked by poor tissue organization, necrosis, and high inflammation, indicating inadequate wound repair.
Experiment 11: Biochemical studies
On the 21st day of the wound healing study, the animals were anesthetized, and blood samples were collected via the retro-orbital method to estimate inflammatory markers, including IL-6 and C-reactive protein (CRP).
The biochemical parameters evaluated in the wound healing study provide insights into the inflammatory response during healing, with IL-6 and CRP serving as key markers (Table 5). Elevated levels of IL-6, a pro-inflammatory cytokine, and CRP, an inflammation marker, typically signal intense or chronic inflammation, which can delay healing.
Co-loaded group had showed balanced wound healing with moderate inflammation, reflected in IL-6 and CRP levels. Notably, the co-loaded scaffold exhibited the highest CRP level among treated groups (4.0 mg/L) but was much lower than the negative control (12.11 mg/L). These results suggest that the combination of curcumin and ß-sitosterol effectively mitigates excessive inflammation, promoting faster healing through the anti-inflammatory properties of curcumin and wound-healing effects of ß-sitosterol.
Curcumin-loaded scaffolds displayed the lowest IL-6 levels (25.65 pg/mL), showing curcumin's strong anti-inflammatory effect. While IL-6 was reduced, CRP levels were slightly higher than in the BS-loaded group, indicating a moderate systemic inflammatory response that still supported effective healing.
ß-sitosterol-loaded scaffolds reported the lowest CRP levels (2.70 mg/L), suggesting reduced systemic inflammation. However, the IL-6 level was higher (33.25 pg/mL), indicating less effective control of local inflammation compared to curcumin alone.
Positive Control Group had shown an Moderate IL-6 (29.55 pg/mL) and CRP (3.61 mg/L) levels indicate a typical healing response without targeted intervention.
The negative control had markedly elevated IL-6 (70.21 pg/mL) and CRP (12.11 mg/L) levels, highlighting excessive inflammation and poor wound healing outcomes.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
, Claims:We Claim,
1. A bioactive, biodegradable hydrogel scaffold composition comprising of:
a) curcumin in an amount of 25 to 50 mg, providing anti-inflammatory and antioxidant properties;
b) beta-sitosterol in an amount of 50 to 100 mg, supporting wound healing and skin repair;
c) chitosan in an amount of 1.45% w/v, dissolved in acetic acid in an amount of 2% v/v, forming a stable polymer base;
wherein the scaffold is characterized by:
i. a porosity of 76.73%, with a drug content of 88.46 ± 1.7% for curcumin and 91.23 ± 3.8% for ß-sitosterol;
ii. a water vapor transmission rate of 69.93 ± 1.2 g/m²h;
iii. a drug release of 30.84% at 12 hours;
iv. achieving 95% wound closure by day 15 and nearly 100% by day 17;
v. promoting wound healing through the sustained release of curcumin and ß-sitosterol.
2. A method for preparing the bioactive, biodegradable hydrogel scaffold, comprising the steps of:
a. dissolving 1.45% w/v chitosan in 2% v/v acetic acid under magnetic stirring at 500 rpm to form a homogeneous polymer solution;
b. dispensing the appropriate amounts of curcumin and beta-sitosterol into 10g of the chitosan solution to ensure even drug distribution;
c. magnetically stirring the mixture until a fully homogeneous solution is achieved, ensuring uniform incorporation of the drugs within the hydrogel matrix;
d. pouring the homogeneous mixture into petri dishes and freezing at -50°c for 20 hours to solidify the hydrogel scaffold structure; and
e. freeze-drying the frozen hydrogel at -50°c for 24 hours to create a porous and stable scaffold structure, suitable for controlled drug release.
3. The method for preparing the bioactive, biodegradable hydrogel scaffold, as claimed in claim 2, wherein the wherein curcumin is present in an amount ranging from 25 to 50 mg per 10 g of the hydrogel.
4. The method for preparing the bioactive, biodegradable hydrogel scaffold, as claimed in claim 2, wherein beta-sitosterol is present in an amount ranging from 50 to 100 mg per 10 g of the hydrogel.
5. The method for preparing the bioactive, biodegradable hydrogel scaffold, as claimed in claim 2, wherein the chitosan solution is magnetically stirred at 500 rpm for a period of 30 minutes to form a uniform polymer solution.

Dated this 19 November 2024

Dr. Amrish Chandra
Agent of the applicant
IN/PA No: 2959

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